PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1

Abstract

In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.

Figure 1. Establishment of a culture-and-replate protocol to investigate mechanisms of regenerative axon growth in vitro

(a) Schematics of the culture-and-replate protocol. DRG neurons were dissociated from adult mice and cultured for 3 days. Neurons were then replated to initiate axon growth anew. Regenerative axon growth was assessed by measuring axon length from replated neurons at 20 hr after replating. (b) In vitro culture recapitulates peripheral axotomy-induced upregulation of several proteins that are encoded by well-known regeneration-associated genes (RAGs), such as ATF3, c-Jun, GAP-43, GADD45a, and SRPP1a. Shown are representative immunoblots from DRG neuronal lysates cultured for either 3 hr or 3 days. Actin antibodies were used as a loading control. (c) DRG neurons grow long, sparsely branched axons after replating, similar to neurons that were conditioned in vivo by sciatic nerve transection. Note that the mode of axon growth after replating is distinct from that induced by nerve growth factor (NGF, 50 ng/ml), which stimulates extensive branching, but relatively modest lengthening. Scale bar, 200 μm. (d, e) Adult DRG neurons were dissociated, cultured for 3 days, and then replated to induce axon growth anew, as depicted in a. Neurons were treated with DBR (20 μM), nocodazole (50 nM), or vehicle control (DMSO) either before (red bars in e) or after replating (blue bars in e), as indicated. Schematics (upper left insets in d) depict when the neurons were treated with drugs of interest. Colored bars indicate the period when neurons were exposed to drugs. White, grey and black arrowheads indicate the starting point of the initial 3-day-culture period, the time of replating, and when neurons were fixed for the analysis, respectively. Regenerative axon growth was assessed by measuring axon length after replating. Representative images neurons after replating are shown in d. Quantification of axon length from three independent experiments is shown in e. Scale bar, 200 μm. Error bars represent s.e.m. *** p < 0.001, unpaired two-tailed student t test. Original immunoblot images are shown in Supplementary Fig. S10.

Figure 2. Activation of PI3K signalling is essential for augmentation of axon growth potential

(a, b) Adult DRG neurons were cultured as depicted in Fig. 1a. Neurons were treated with LY294002 (LY, 10 μM), U0126 (20 μM), or vehicle control (DMSO) either before (red bars in b) or after replating (blue bars in b), as indicated. Regenerative axon growth was assessed by measuring axon length after replating. Representative images of replated neurons are shown in a. Quantification of axon length from three independent experiments is shown in b. Scale bar, 200 μm. Error bars represent s.e.m. *** p < 0.001, unpaired two-tailed student t test. (c) Adult DRG neurons from bax^−/− mice were cultured as depicted in Fig. 1a. Neurons were treated with LY294002 (LY, 10 μM) or vehicle control (DMSO) either before (red bar) or after replating (blue bar), as indicated. Regenerative axon growth was assessed by measuring axon length after replating. Quantification of axon length from three independent experiments is shown. Error bars represent s.e.m. *** p < 0.001, unpaired two-tailed student t test. (d) Validation of the dominant-negative PI3K construct (dnPI3K). Representative immunoblots from 3-day-cultured DRG neurons transfected with either dnPI3K or a control vector (EGFP). (e, f) Dissociated adult DRG neurons were transfected in vitro with EGFP alone (control) or with dnPI3K (myc tagged). Neurons were cultured for 3 days, followed by replating to initiate axon growth anew. Neurons were fixed at 20 hr after replating and stained for neuronal tubulin βIII, TuJ1 (red) or the myc tag. Transfected neurons are shown in green (EGFP or myc positive neurons). Representative images of replated neurons are shown in e. Quantification of axon length from three independent experiments is shown in f. Scale bar, 100 μm. Error bars represent s.e.m. ** p < 0.01, unpaired two-tailed student t test. (g) Representative immunoblots of phosphorylated Akt and ERK1/2 detected in DRGs from naïve or injured mice that were subjected to sciatic nerve transection. (h) Representative immunoblots of time-course analysis of AKT phosphorylation in DRGs in response to peripheral nerve injury. Original immunoblot images are shown in Supplementary Fig. S10.

Figure 3. PI3K signalling is required for activating proregenerative responses and subsequent axon regeneration in vivo

(a) Schematics of in vivo electroporation and investigation of axon regeneration. (b, c) L4/L5 DRGs of an adult mouse were electroporated in vivo with EGFP alone (control) or together with dominant-negative PI3K (dnPI3K) at a ratio of 1:3, followed by the sciatic nerve crush as depicted in a. The lengths of all identifiable regenerating axons in the whole mount nerves were measured from the crush site (arrowheads) to the distal axon tips. Lengths of total of 120 axons from six control mice and 124 axons from six dnPI3K-transfected mice were measured. Quantification of axon length is shown in b, and representative images are shown in c. Scale bar, 500 μm. Error bars represent s.e.m. *** p < 0.00001, unpaired two-tailed student t test. (d) L4/L5 DRGs in adult mice were electroporated in vivo with EGFP (control) or dnPI3K (myc tagged), immediately followed by sciatic nerve transection. At 7 days after sciatic nerve transection, neurons from L4/L5 DRGs were cultured overnight and stained for neuronal tubulin, TuJ1 (red) or the myc tag. Transfected neurons are shown in green (EGFP or the myc positive). Experimental scheme is depicted in the upper left. Representative images (right) and quantification of axon length (lower left) from three independent experiments are shown. Scale bar, 100 μm. Error bars represent s.e.m. *** p < 0.001, unpaired two-tailed student t test. (e) Adult mice were first subjected to sciatic nerve transection. At 7 days after the nerve injury, L4/L5 DRGs were electroporated in vivo with EGFP (control) or dnPI3K (myc tagged). 1 day after the in vivo electroporation, neurons from L4/L5 DRGs were cultured overnight and stained for neuronal tubulin, TuJ1 (red) or the myc tag. Transfected neurons are shown in green (EGFP or the myc positive). Experimental scheme is depicted in the upper left. Representative images (right) and quantification of axon length (lower left) from three independent experiments are shown. Scale bar, 100 μm. Error bars represents s.e.m. *** p < 0.001, unpaired two-tailed student t test.

Figure 4. Regenerative axon growth from adult sensory neurons requires mTOR-independent protein synthesis

(a, b) Adult DRG neurons were dissociated, cultured for 3 days and then replated to initiate axon growth anew, as depicted in Fig. 1a. Neurons were treated with rapamycin (20 nM), cycloheximide (5 μg/ml), or DMSO (control) either before (red bars in b) or after replating (blue bars in b), as indicated, and axon length was measured at 20 hr after replating. The schematics and symbols (upper left insets in a) are identical to Fig. 1d. Representative images of replated neurons are shown in a, and quantification of axon length from three independent experiments is shown in b. Scale bar, 200 μm. Error bars represent s.e.m. n.s., statistically insignificant difference, unpaired two-tailed student t test. (c) Representative images of control (upper panels) and rapamycin-treated (lower panels) adult DRG neurons immunostained with TuJ1 and phospho-S6 antibodies. Note that rapamycin treatment completely prevented S6 phosphorylation, but had no effect on axon growth. Scale bar, 200 μm. (d) Representative immunoblots from adult DRG neurons cultured for 3 days in the presence or absence of rapamycin. Actin antibodies were used as a loading control. Note that rapamycin treatment completely prevented S6 phosphorylation. Original immunoblot image is shown in Supplementary Fig. S11.

Figure 5. Inactivation of GSK3 in the cell body is responsible for augmentation of transcription-dependent axon growth potential downstream of PI3K signalling

(a) Representative immunoblots of phosphorylated GSK3α-Ser21, GSK3β-Ser9 and CRMP2-Thr514 detected in L4/5 DRG lysates from naïve or injured mice that were subjected to sciatic nerve transection. (b) Representative immunoblots of phosphorylated GSK3β-Ser9 detected in dissociated adult DRG neurons cultured for either 3 hr or 3 days. For the 3-day cultures, neurons were treated with LY294002 (LY, 10 μM) or vehicle control (DMSO), as indicated. (c, d) Adult DRG neurons were dissociated, cultured for 3 days, and then replated. Neurons were treated with 6-bromoindirubin-3′-acetoxime (BIO, 500 nM), SB216763 (SB, 10 μM), and/or LY294002 (LY, 10 μM), as indicated, during the initial 3-day-culture period. Replated neurons were cultured in the absence of drugs. Axon length was measured at 20 hr after replating. Representative images of replated neurons are shown in c, and quantification of axon length from three independent experiments is shown in d. Scale bar, 200 μm. Error bars represent s.e.m.** p < 0.01; *** p < 0.001, unpaired two-tailed student t test. (e, f) Adult DRG neurons were loaded in the somal side of the two-compartment chambers as described in Methods. LY294002 (LY, 10 μM) and/or BIO (500 nM, GSK3i) was locally applied either in the somal (red bars in f) or the axonal side (blue bar in f), as indicated. Y-axis in f depicts the number of microchannels occupied by axons exiting the channels and entering the axonal side (after normalization against the total number of channels). Representative images (e) and quantification (f) of axons entering the axonal compartment are shown. Schematics of the two-compartment chambers (upper left corner) depict where drugs were added. Treatment to the axonal and the somal side is illustrated in blue and red, respectively. Scale bar, 200 μm. n=3, error bars represent s.e.m. * p < 0.01 compared to control; *** p < 0.001; n.s. statistically insignificant difference compared to control, unpaired two-tailed student t test. Original immunoblot images are shown in Supplementary Fig. S11.

Figure 6. β-catenin is dispensable for sensory axon regeneration

(a) Representative immunoblots of DRG lysates from naïve or injured mice. To induce injury, mice were subjected to sciatic nerve transection and DRGs were collected after 3 days. Actin antibodies were used as a loading control. Quantification of β-catenin level normalized against actin is presented (right). (b) Representative immunoblots from DRG neurons cultured for either 3 hr or 3 days. For the 3 day cultures, neurons were grown in the presence of LY294002 (LY, 10 μM) or vehicle control (DMSO), as indicated. Actin antibodies were used as a loading control. Quantification of β-catenin level normalized against actin is presented (right). Error bars represent s.e.m. n.s., statistically insignificant difference, unpaired two-tailed student t test. (c) Representative blots for validating the efficacy of the siRNA against β-catenin (si-β-catenin) after in vivo electroporation into DRGs. Actin antibodies were used as a loading control. (d, e) L4 and L5 DRGs of an adult mouse were electroporated in vivo with either EGFP alone (control) or together with si-β-catenin, followed by sciatic nerve crush at 2 days after the electroporation. Axon regeneration was assessed by whole-mount analysis at 3 days after nerve injury. In vivo electroporation and investigation of axon regeneration were performed as depicted in Fig. 3a. Using whole-mount nerve segments, the lengths of all identifiable regenerating axons were measured from the crush site (arrowheads) to the distal axon tips. For quantification, lengths of a total of 145 axons from six control mice and 140 axons from six si-β-catenin-transfected mice were measured. Quantification (d) and representative images of axon regeneration (e) are shown. Scale bar, 500 μm. Error bars represent s.e.m. Original immunoblot images are shown in Supplementary Fig. S10.

Figure 7. Induction of Smad1 downstream of the PI3K-GSK3 pathway is essential for augmentation of the transcription-dependent axon growth potential

(a, b) Western blot analysis of DRG neurons cultured for 3 hr or 3 days to examine the levels of phospho-Smad1 (p-Smad1), Smad1 or GAP43. For the 3 day cultures, neurons were treated with DMSO, LY294002 (LY, 10 μM) and/or 6-bromoindirubin-3′-acetoxime (BIO, 500 nM). Representative images and quantification of Western blot analysis are shown in a and b. n=3, error bars represent s.e.m. * p < 0.05; *** p < 0.001, unpaired two-tailed student t test. (c, d) Dissociated adult DRG neurons were transfected in vitro with EGFP, Smad1 (flag tagged) and/or dominant-negative PI3K (dnPI3K) (myc tagged). Neurons were cultured for 3 days and then replated for overnight culture. Neurons were then fixed and stained for neuronal tubulin, TuJ1, the flag tag, or the myc tag. Representative images of replated neurons are shown in c, and quantification of axon length from three independent experiments is shown in d. Scale bar, 100 μm. Error bars represent s.e.m. * p < 0.05; ** p < 0.001, unpaired two-tailed student t test. (e) Dissociated adult DRG neurons were transfected with EGFP alone (control) or together with siRNAs against Smurf1 (siSmurf1) and/or dnPI3K, as indicated, and cultured for 3 days. Neurons were then replated and culture overnight. Quantification of axon length from three independent experiments is shown. Error bars represent s.e.m. * p < 0.05, unpaired two-tailed student t test. Efficacy of siSmurf1 was validated in adult DRG neurons (immunoblots in upper panel). (f) Adult DRG neurons cultured for 3 hr or 3 days were subjected to quantitative real time polymerase chain reaction (qRT-PCR) to examine the mRNA level of smad1 or gap43. For the 3-day cultures, neurons were treated with DMSO, LY294002 (LY, 10 μM) and/or BIO (500 nM), as indicated. Values were normalized against the mRNA level of the DMSO-treated sample. n=3, error bars represent s.e.m. * p < 0.05; *** p < 0.001, unpaired two-tailed student t test. Original immunoblot images are shown in Supplementary Fig. S12.

Figure 8. Acute depletion of Smad1 in vivo prevents sensory axon regeneration

(a) Representative immunoblots of DRG lysates from naïve or injured mice. Mice were subjected to sciatic nerve transection to induce injury. DRGs were collected at 3 days after injury and subjected to Western blot analysis. Representative immunoblots for phospho-Smad1 (p-Smad1) and Smad1 are shown. Actin antibodies were used as a loading control. (b, c) L4 and L5 DRGs of an adult mouse were electroporated in vivo with either EGFP alone (control) or together with siRNAs against Smad1 (siSmad1), as indicated, followed by sciatic nerve crush at 2 days after the electroporation. Axon regeneration was assessed by whole-mount analysis at 3 days after nerve injury. In vivo electroporation and investigation of axon regeneration were performed as depicted in Fig. 3a. Using whole-mount nerve segments, the lengths of all identifiable regenerating axons were measured from the crush site (marked by epineural suture, arrowheads) to the distal axon tips. For quantification, lengths of a total of 92 axons from six control mice and 121 axons from six siSmad1-transfected mice were measured. Quantification (b) and representative images (c) of regenerating axons are shown. The inset shows knocking down of Smad1 by siSmad1 in adult DRG neurons in vivo. Scale bar, 500 μm. Error bars represent s.e.m. * p < 0.05, unpaired two-tailed student t test. Original immunoblot images are shown in Supplementary Fig. S12.